METHOD FOR OBTAINING MALE-STERILE PLANTS

Abstract: A gene isolated from the cDNA library of a plant which encodes a protein MS2 involved in pollen development, said gene having the nucleotide sequence shown in Fig. 2 or a homologous gene. Also disclosed is a promotor of said gene having the nucleotide sequence shown in Fig. 3 or a homologous promotor. Further the invention discloses a recombinant polynucleotide which can be suitably used for obtaining a male-sterile plant, essentially comprising a) an inhibitor gene capable of inhibiting the expression of a target gene encoding the MS2 protein or a homologous target gene, and b) a promotor that is active in the tapetum of said plant.

Description: METHOD FOR OBTAINING MALE-STERILE PLANTS FIELD OF THE INVENTION The invention involves genetic manipulation of plants by the application of recombinant DNA technology. The invention describes a method for obtaining plants which display nuclear encoded male-sterility, due to expression of said recombinant DNA as well as parts of said plants which are either sexually or asexually reproducible, or both. Background of the art Heterosis is the effect that progeny obtained through cross- pollination show better agronomical properties than progeny derived through self-pollination. These better properties result in a higher profit which is the reason that the development of hybrid seed is one of the prime objectives for the seed industry. To obtain hybrid seeds it is necessary that the female plants are unable to self-pollinate. Many crop plants accommodate both the female and male reproductive organs on the same individual and thus self-pollination is dominating cross-pollination. Introduction of male sterility in many crops is thus necessary for hybrid seed production. In production fields the male-sterile acceptor and male- fertile donor plants are grown side by side and after cross- pollination the hybrid seed is harvested. Hybrid seed is separately collected from the non-hybrid seeds formed on the donor plants by destruction of these male-fertile plants before the harvest. This approach makes the discrimination between fertile and sterile plants necessary which can be accomplishes j ~ >y ι_.u.e appearance OJ_ v___. j.___ ent ϋ.s otypes. niso a selectable marker gene such as a gene coding for a herbicide resistance, closely linked to the male-sterility locus can be used for this purpose. Selection of the hybrid seeds can than be accomplished by spraying the herbicide, which will result in the destruction of the male-fertile plants . Alternatively the hybrid seeds can be selected after harvesting by a phenotypic marker expressed at the seed level. Nowadays in agricultural practice male-sterile parental lines are obtained by physically emasculation of the plants or by the application of cytoplasmic or nuclear encoded male-sterile mutants. There are only a limited number of natural male-sterile mutants available in the commercial interesting crops. This renders the latter approach often not feasible. In addition naturally male-sterile plants have their disadvantages . Their preparation is laborious and their maintenance and propagation difficult. They often show additional, detrimental traits and a difficult inheritance. The development of nuclear encoded male-sterile plants by the application of recombinant DNA techniques has succeeded in new approaches which circumvent most of the disadvantages mentioned above. STATE OF THE ART The International Patent Application 092/18625, MOGEN International N.V. proposes methods for the production of restorable male-sterile plants in general terms, essentially comprising integration of a recombinant polynucleotide into their genome, essentially comprising an inhibitory gene, which, upon proper expression in the anthers of the plant, is capable of inhibiting expression of one or more genes encoding an enzyme involved in the synthesis of chalcone, or one of its precursors. DEFINITIONS Antisense gene: a nucleotide sequence having a homology of more than 50%, preferably more than 80% with a target gene as defined herein, and which is linked to a promotor in 3' to 5' orientation with respect to the target gene and can be expressed as an RNA molecule. Gene or sense gene: a nucleotide sequence that can be expressed as RNA molecule and/or polypeptide. Promotor: a nucleotide sequence which directs the expression of a (sense-) gene or antisense gene, or nucleotide sequences derived thereof. Inhibitor gene: a (sense-)gene or antisense gene, expression of which leads to prevention or inhibition of the expression of a target gene as defined herein. Repair gene: a gene capable to prevent or sufficiently inhibit the action of an inhibitor gene thus repairing the activity of a target gene. Target gene. a gene which activity is to be inhibited by proper expression of an inhibitor gene as herein defined. SUMMARY OF THE INVENTION The present invention describes the male-sterile Arahidopsis thaliana mutant PA and the molecular isolation and analysis of the gene, called MS2 (Fig. 1) . Mutation of this gene is responsible for the male-sterile phenotype of PA. The only other male sterile mutant of Arabidopsis thaliana , msl, has been reported by Van der Veen and irtz (1968) . This mutant was obtained by classical mutagenesis and is the only other male sterility mutant described in the literature, which does not show pleiotropic effects. Further molecular characterization of msl has not been reported so no information is available on the nature of the male-sterility gene. Accordingly, the present invention provides a gene isolated from the cDNA library of a plant which encodes a protein indicated as MS2 which is involved in pollen development, said gene having the nucleotide sequence as shown in Fig. 2 or a homologous gene. Further, this invention provides a promotor of the MS2 gene isolated from the chromosomal DNA of a plant, said promotor having the nucleotide sequence as shown in Fig. 3 or a homologous promotor. Further, the present invention provides recombinant polynucleotides which can be suitably used for obtaining a male-sterile plant, essentially comprising: a) an inhibitor gene capable of inhibiting expression of a target gene in the said plant, encoding the MS2 protein involved in pollen development said gene having the sequence as shown in Fig. 2 or a homologous target gene, and b) a promotor, that is active in the tapetum of said plant, linked to said inhibitor gene as to achieve expression thereof in the tapetum of said plant. In the preferred embodiment of the invention the inhibitor gene is a (sense-) gene or an antisense gene directed against the target gene. In another preferred embodiment according to the invention the promotor that is active in the tapetum of a plant comprises the promotor of the MS2 gene, said promotor having the sequence as shown in Fig. 3. The present invention also provides a method for obtaining a male-sterile plant comprising the steps of: a) transferring a recombinant polynucleotide according to the invention to cells of a male-fertile plant, b) generating whole new plants from cells having incorporated said recombinant polynucleotide, and c) selecting a plant that is male-sterile. Yet another embodiment of the invention is a recombinant plant genome, comprising incorporated therein a recombinant polynucleotide according to the invention. In a yet further preferred embodiment of the invention a method is provided for a cost effective production of hybrid seed, due to use of large numbers of heterozygous male-sterile plants, which have been obtained by crossing a homozygous male-sterile plant of the desired variety with a male-fertile plant of the same variety. The invention further encompasses (hybrid) seed obtained through crossing or selfing of any of the plants according to the invention. Other preferred embodiments of the invention are the plasmids pMS2 and pMS2as (see figure 6). DESCRIPTION OF THE FIGURES Figure 1: Southern analysis .of the progeny of a cross between the male-sterile Arabidopsis thaliana mutant PA and male-fertile Arabidop¬ sis thaliana ecotype Landsberg erecta. Figure 2: The nucleotide sequence of the MS2 mRNA. Figure 3: The nucleotide sequence of the promoter of the MS2 gene. Figure 4a: Northern analysis of the expression of the MS2 gene in different organs of Arabidopsis thaliana . Figure 4b: Northern analysis of the expression of the MS2 gene in different organs of Raphanus satlr ε (radish) . Figure 5: Southern analysis of the presence of MS2 homologues in radish and oilseed rape. Figure 6: A diagrammatic representation of the different cloning steps for obtaining the chimeric sense and antisense MS2 constructs and plasmids pMS2 and pMS2as. DETAILED DESCRIPTION OF THE INVENTION 1. Isolation of the MS2 gene of Arabidopsis thaliana The maize Enhancer-Inhibitor (En-I) (Peterson, 1953), also known as Suppressor-Mutator (Spm) (McClintock, 1954), transposable element system has been used for transposon tagging of a number of genes in maize and was shown to transpose when introduced by transformation in a heterologous plant species (Masson and Federoff, 1987; Pereira and Saedler, 1989; Frey et al., 1989). We constructed a transformation vector containing three units, namely: a) an immobile En transposase source (Masson and Federoff, 1987), under control of the strong Cauliflower Mosaic Virus 35S promotor; b) a mobile I element as insertion mutagen (Schwartz-Sommer et al., 1985); c) a hygromycin phosphotransferase gene (Van den Elzen et al., 1985) conferring resistance to the antibiotic hygromycin. This 'in cis two element En-I' vector construct was used for AgrroJacterium tumefaciens mediated transformation of Arabidopsis thaliana (Valvekens, 1988) . A male sterile plant was found among the third generation (T3) progeny of a successively self-fertilized primary transformant (Tl) with frequently transposing I elements. The plant was coded PA (Pollen Absent) . As the mutation involved can be complemented by msl, the only other male-sterile mutant without pleiotropic effects described in Arabidopsis (Van der Veen and irts, 1968) , we called this novel mutant ms2. Segregation analysis of the male-sterile phenotype and the I transposable element was performed as follows . DNA of F2 progenies from a cross PA x Landsberg erecta was Southern transferred and hybridized with an I element specific probe (figure 1) . DNA was digested with Hindlll which does not cut inside the I element. This F2 progeny lacked the En transposase genes and therefore all detected fragments denote stable I element inserts. A 6.6 kb I element containing fragment (■) , is homozygous only in plants displaying a male sterile (ms) phenotype. Plant F2 3-1 (*) has only this ms2 accompanying I element and was therefore used for the isolation of the transposon flanking DNA by Inverse Polymerase Chain Reaction (IPCR) (Masson and Federoff, 1989; Ochman et al. 1988) . The IPCR fragment was cloned, sequenced and used as a probe to isolate clones from a flower specific cDNA ( eigel et al . , 1992) as well as a genomic lambda library of Arabidopsis . Analysis of the MS2 mRNA The cDNA clone (largest out of 3 isolated) was double- stranded sequenced using the Taq Dye Deoxy Terminator Cycle Sequencing Kit (Applied Biosystems) and an automated DNA sequencer (Applied Biosystems) . Nucleotide and predicted amino-acid sequences of MS2 cDNA are shown in figure 2. Sequence analysis was performed using the Genetics Computer Group (GCG) software package (Devereux et.al. 1984) . Nucleotides are numbered beginning with the first position of the ATG initiation triplet. A nonsense codon upstream (-36) indicates that this is the translation start. The polyadenylation signal sequence (position 1967 underlined) precedes the poly(A) tail. The position of the I-element insertion (AAA) at position 1800 is underlined. The last three amino acids (Gly-Arg-Gly) contain a putative C-terminal microbody targeting signal (CMTS) suggesting a role of this part in the protein which is absent in male sterile mutant alleles due to excision footprints causing frameshifts. Data base searches (FASTA/BLAST) revealed homology to a DNA sequence (EMBL release 33.0, 12/92) located upstream of the wheat mitochondria 26S ribosomal RNA rrn26 gene. The region of homology in the MS2 cDNA (1040- 1203) is underlined and shows 77.6% identity to the wheat mitochondrial nucleotide sequence. This region is located about 2 kbp upstream of the rrn26 gene at position 650-814 of the published sequence (Spencer et al., 1992). No function has been ascribed to this mitochondrial DNA segment which reveals an open reading frame whose predicted amino acids show 81.7% identity to the MS2 protein (55 amino-acids). The homology in the mitochondrial DNA segment does not extend beyond this region but is precisely flanked by intron-exon boundaries. Fig. 3 shows the nucleotide sequence of the promotor of the MS2 gene. Said sequence is obtained from A. thaliana ecotype Landsberg erecta geno ic clone of the MS2 gene. The TATA box of the MS2 gene is shown underlined (position 835) and the 5' end of the MS2 cDNA sequence is marked with an asterisk (position 957). A unique Asel restriction site (ATTAAT) at position 953 allows cloning of the promotor for further plasmid constructions described. 3. Analysis of the expression of the MS2 gene In Arabidopsis thaliana . Northern analysis of the expression of the MS2 gene in different organs of Arabidopsis thaliana showed that MS2 mRNA could only be detected in flowers and siliques but not in seedlings and leaf or stem tissue (see figure 4a). As shown in Figure 4b MS2 RNA could also be detected in flowerbuds of radish. By Southern blot analysis we have confirmed that MS2 homolo¬ gous genes are present in radish but also in oilseed rape (Fig 5) which indicates that male sterility can be induced in oilseed rape by sense-MS2 or antisense-MS2 expression. 4. The present invention envisages the following steps towards obtai¬ ning male-sterile plants: A sense-MS2 or antisense-MS2 gene of Arabidopsis thaliana or its homologous counterpart of other species is placed under the control of the MS2 promotor, which DNA sequence is shown in figure 3, and these constructs can be used for the transformation of fertile crop plants. After selection of transformed plants which express the construct, and after the plants are allowed to flower, transgenic plants showing disturbed pollen development can be selected. The diagram of Fig. 6 shows the steps of the process for preparing the above sense and antisense constructs. A binary vector (origin pBIN19), containing a chimaeric NPT II gene conferring kanamycin resistance to plant cells, with unique Sail (S) and EcoRI (R) restriction sites serves as the basic vector unit. The MS2 gene promotor and CaMV 35S poly (A) addition signal/terminator (term) are cloned after Klenow polymerase treatment (Klen) . The MS2 cDNA fragment is made blunt (with Klenow polymerase) and cloned into the Smal restriction site in two orientations with respect to the MS2 promotor (prom) . This results in the two plasmids designated pMS2 (sense) and pMS2as (anti-sense). Other abbreviations used: RB= T-DNA right border; LS= T-DNA left border; 5' and 3' refer to the ends of the cDNA (coding strand) defined by unique restriction sites in the cDNA clone. In general nuclear encoded male-sterile plants can thus be obtained and used for hybrid seed production. Plants of a selected variety have to be genetically transformed by introducing into cells of the said plant one or more recombinant polynucleotides, essentially comprising one or more inhibitor genes, which upon proper expression in the plant, are capable of inhibiting expression of a MS2 gene. Inhibition of the expression of the MS2 gene will result in male-sterile plants. This can be accomplished by proper expression of an inhibitor gene directed against that target gene. Inhibitor genes can be suitably selected from a range of alternatives, including homologous or heterologous (i.e obtained from a different source) sense and antisense (synthetic) -genes or parts thereof with a suitable length and homology for proper inhibition, as illustrated in International Patent Application 092/18625, MOGEN International NV and International Patent Application O90/11682, DNA Plant Technology Inc. Preferably the inhibitor gene is expressed in the tapetum according to the present invention. This can be accomplished by fusing the inhibitor gene under control of the promotor derived from the MS2 gene (see figure 4) from Arabidopsis or its heterologous counterpart (i.e obtained from another source) . Transfer of recombinant polynucleotides into plants or parts thereof can be achieved by numerous techniques. Some of them are listed here as illustration and comprise transformation of protoplasts using the calcium/polyethylene glycol method (Krens et al . 1982; Negrutiu et al. , 1987) , electroporation (Shillito et al . , 1985) , microinjection (Crossway et al . , 1986) , (DNA or RNA coated) particle bombardment (Klein et al., 1987) , infection with viruses and the like, natural DNA transfer by Agrobacterium species preferably by the use of the so-called binary vector system (Bevan et al . , 1984) . After identification of transformed plant material, whole plants are regenerated using well-known protocols described in literature (vide e.g. Horsch et al. , 1985) . There does not exist any restriction towards parts of the plants used. After transformed plants have been obtained, they can be evaluated for the presence of the desired trait and/or the degree to which the desired traits are expressed. A first evaluation may include the level of expression of the inhibitor gene and the extent to which the transgenic plants are male-sterile. Subsequently transgenic plants can be selected that show stable and/or predictable inheritance of the male-sterile trait, and the like. Next the (heterozygous) male sterile plants can be used directly for the production of hybrid seed, or alternatively be selfed with rescued pollen in order for obtaining homozygous male- sterile plants. Clearly, the advantage of having a few homozygous male-sterile plants permits one to rapidly acquire large amounts of heterozygous male-sterile seed which can be directly used for large scale production of hybrid seed. The present invention can be applied in any plant capable of self-pollination, for which the production of hybrid seeds is of commercial interest. The invention also provides a method for obtaining homozygous male-sterile plants. This can be accomplished by selfing the heterozygous male-sterile plants according to the invention. The development of pollen of these plants can be rescued by overcoming the inhibition of in vivo MS2 protein synthesis. Inhibition of MS2 gene expression can be circumvented by allowing (temporary) in vivo production of MS2 protein in the tapetum of the plant. This can be achieved by introducing in addition to the inhibitor gene, a repair gene under control of an inducible promotor. In this configuration the expression of the repair gene can be externally controlled by adding an appropriate inducer. Preferably such a repair gene includes a gene at least containing part of the MS2 gene, that is capable upon expression of inhibiting the effect of expression of the antisense MS2 gene. Alternatively other approaches to inhibit the expression of an inhibitor gene as described in International Patent Application 092/18625, MOGEN International N.V. , can be used. If hybrid crops are grown for their seed or fruit, inducible restoration of fertility is necessary. Male-sterility must then be eliminated to permit pollination and harvest of seed or fruit. Repairing male-fertility may be invoked by administration of an inducer to the crop in the field, causing a sufficiently high expression of the repair gene to neutralize the inhibition of the MS2 gene by the inhibitor gene. ADVANTAGES Former methods to provide nuclear male sterile plants involve expression of genes which encode products that are toxic to somatic cells, such as DNAses, RNAses, proteases or one of the enzymes involved in the chalcon biosynthesis pathway. Chalcon is a key compound in the flavonoid biosynthesis pathway. Flavonoids are secondary metabolites that are known to have a key-function in the pigmentation of flowers and fruit. In addition flavonoids appear to be involved in defense against phytopathogens (Lamb et al. , 1989) , the protection against UV-light (Schmelzer et al . , 1988) and the induction of nodulation (Long et al . , 1989) . Flavonoids have also been implicated in the regulation of auxin transport (Jacobs and Rubery, 1988) and resistance to insects (Hedin and Waage, 1986) . As a consequence of the properties mentioned above of the toxic products or the role of the product of the inhibited gene in the plant, strict developmental and tissue-specific expression limited to anthers only is required. The present method does not necessarily require such a strict developmental or tissue-specific expression. Male-sterile plants according to the invention are highly sterile and appear entirely female-fertile. The present invention also provides methods for obtaining homozygous male-sterile plants. Only a few are needed which can be multiplied in vitro by well-established techniques. The homozygous male-sterile acceptor plant is cross- fertilized with the male-fertile donor plant, and the heterozygous male-sterile seed used for hybrid crop production. Of the hybrid seed produced, 50% will be male- sterile and 50% male-fertile. In case this ratio is not sufficient for obtaining high yield of commercial product (i.e seed or fruit) by self-fertilization, this ratio can be ameliorated by partial restoration of male-sterility by administration of an inducer to activate expression of the restorer gene. EXPERIMENTAL Construction of plasmid cwEnN::! The plasmid cwEnN::I used for AgrroJbacterium tumefaciens transformation was constructed with the aim of designing a phenotypic excision assay using kanamycin resistance, for the En-I transposon family, which can be used in a number of plant species. The binary vector pGDW3.1 (Wing et al. , 1989) was the source of the chimeric nopaline synthase inos) promoter-hygromycin phosphotransferase (HPT) gene used for selection during transformation. A chimeric neomycin phosphotransferase II (NPTII) gene was first inserted into pGDW3.1 which is similar to the NPTII gene used by Baker et al . (1987) except that a Clal linker was introduced 5' to the unique BamHI present in the untranslated leader between the T R 1' promoter and the NPTII codogenic region. This introduced a translation start (GCGATGG) 5' to the BamHI site. In addition, the NPTII translation start was removed by exchanging the NPTII gene sequences from plasmid pBCKl (Kaulen et al . , 1984) downstream of the BamHI site. This generated the plasmid pBHN in which a translation start is present upstream of the unique BamHI site into which an I element, 1- 6078 of 2.2 kb with 56 bp flanking DNA (Pereira and Saedler, 1989) , was inserted to generate plasmid pBHNI. En - 1 (Pereira et al. , 1986) was digested with BssHII, which cuts at positions 399 and 8041, made blunt with the Klenow fragment of DNA polymerase I and cloned between the cauliflower mosaic virus (CaMV) 35S promoter-terminator cassette, originating from pD51 (Pietrzak et al. , 1986) in pBR322. This clipped-wing En (cwJEn) under control of the 35S promoter was next cloned into the binary vector pBHNI to produce plasmid cwEnN::I (Figure 1) which was mobilized into the Agrrobacterium strain pGV3101 (pMP90RK) (Koncz and Schell, 1986) . Plant transformation, propagation and phenotypic excision assay For plant transformation, root explants of ecotype Landsberg erecta were infected with the Agrobacterium strain containing the binary vector construct, according to a method described by Valvekens et al. (1988). Transformed calli were selected on medium containing hygromycin at 20 mg.l "1 . Emerging T, shoots were grown without selection and allowed to self-fertilize and set seed in vi tro . Seedset of A . thaliana is very poor in vi tro due to high humidity. This was solved by lifting the lid of the pots a little. Growth conditions were 12 to 16 hrs light at 22°C in growth chambers. Seeds of T 2 and subsequent generations were sterilized in Eppendorf tubes by treatment with ethanol 70% (2 min) , 50% commercial bleach (5% sodium hypochlorite, 5 min) and five subsequent washes with sterile water. The seeds were sown either wet or dry on GM medium (Valvekens et al. , 1988) or half strength MS (Murashige and Skoog, 1962) solidified with 0.8% purified agar. For determining the number of T-DNA loci, seeds were mostly sown on half strength MS with 20 mg.l "1 hygromycin. Using no sugar in the medium permitted sterilization with 70% ethanol for 2 min and only two to three washes with sterile water. Segregation of antibiotic resistance was scored 5 to 10 days after germination. If needed seedlings were transferred to soil (compost:vermiculite:sand=4:l:l) and grown in a climate controlled greenhouse at 20°C with additional light (16 hrs) . To prevent cross-fertilization and spread of seeds, plants were grown in Aracon containers (Beta Developments, Gent, Belgium) . For the phenotypic excision assay seeds were germinated on GM medium containing 100 mg.l" 1 kanamycin sulphate. Resistance, variegation or sensitivity was scored 5 to 10 days after germination. Variegated and sensitive seedlings were transferred to GM without antibiotic to rescue them before transferring to soil. Ploidy number was determined by counting chloroplasts in stomatal guard cells of the lower epidermis of young leaves as described by Detrez et al. (1989) . At least 25 stomata of different T 2 and T 3 plants were regarded. Genomic DNA isolation Genomic DNA was isolated from young rosette leaves of individual greenhouse grown plants for both PCR and Southern analysis. The method we used was a modified version of the one described by Dellaporta et al . (1983) . 100 mg of liquid N 2 -frozen leaf tissue was grinded in a 1.5 ml Eppendorf tube and mixed with 300 μl of extraction buffer (100 mM Tris-HCl pH 8.0, 50 mM EDTA pH 8.0, 500 mM NaCl, 10 mM β-mercapto- ethanol) and 25 μl of 20% SDS . The mix was incubated at 65°C for 10 min, after which 115 μl 5 M potassium acetate was added, mixed and kept on ice for at least 20 min and then centrifuged. The DNA was precipitated from the resultant supernatant by adding 0.6 to 2 volumes of isopropanol and if needed kept at -20°C for a few hours. The pellet was redissolved in 200 μl of TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0) centrifuged for 5 min to remove debris, transferred to a new tube and RNase A added to 20 μg.ml "1 . The reaction mix was incubated at 37°C for 15 min when Proteinase K was added upto 50 μg.ml '1 . After another 15 min at 37°C the solution was phenol/chloroform extracted. DNA was precipitated again by adding 0.25 volumes of 10 M ammonium acetate and 2 volumes of 100% ethanol. The pellet was dissolved in 20 μl of TE. This procedure was either up or down scaled if different amounts of leaf tissue were used. It generally yielded about 1 to 2 μg of DNA per 100 mg leaf tissue. DNA-methodology DNA subcloning, restriction analyses and sequencing were performed using standard procedures well know to persons skilled in the art, vide e.g. Maniatis et al. , 1982. PCR and sequence analysis Inverse PCR analysis to amplify transposon-flanking DNA of transformants was performed with 100 to 200 ng of genomic DNA. For the reaction 15 p ole of each primer was used, 0.1 mM of dNTPs (equal amounts of each dNTP) and 0.1 unit of SuperTaq DNA Polymerase (HT Biotechnology Ltd., Cambridge, England) in a total volume of 50 μl reaction buffer as supplied along with the enzyme (10 mM Tris-HCl pH 9'.0, 50 mM KC1, 0.01% (w/v) gelatin, 1.5 mM MgCl 2 and 0.1% Triton X- 100) . 30 cycles of denaturation (94°C, 30 sec) , annealing (61°C, 60 sec) and extension (72°C, 60 sec) were carried out. One tenth volume of PCR product was checked on agarose gel and if of interest cloned in Bluescript SK + as a blunt ended fragment after Klenow treatment and gel elution. Sequence analysis was performed on double stranded super- coiled plasmid using an automated sequencer (Applied Biosystems) . Initially one strand, if needed both strands were sequenced. Southern blot analysis Genomic DNA was digested with both EcoRI and Bglll and 0.5 to 1 μg was separated on a 0.8 % agarose gel in Tris-acetate running buffer. After electrophoresis the DNA was alkali transferred onto a Gene Screen Plus membrane by vacuum blotting. The blots were prehybridized and hybridi¬ zed following the procedure recommended by the membrane manufacturer. Probes (Figure 1) were [ P] random prime labelled DNA fragments. A 0.27 kb fragment containing the left border of En (upto a Sail re¬ striction site) was used to detect J elements. The T-DNA right border probe was a 1.1 kb fragment containing the HPT gene. A fragment of 1.8 kb containing the complete NPTII gene plus octopine synthase (ocs) terminator was used to detect excision fragments. In the experiment shown in Fig. 5, MS2 cDNA was used as probe. After hybridization the membranes were washed twice with 2xSSC, 1 % SDS at 65 °C for 30 minutes and autoradiographed to X-ray films (Fuji or Kodak) at -80 °C using intensifying screens. Isolation of RNA Plant material was collected, frozen in liquid N, and grinded using mortar and pestle. Equal volumes of extraction buffer (0.1 M Tris-HCl, pH 9.0, 1% SDS, 0.1 M LiCl, 0.01 M EDTA) and phenol were added (4 ml each) and the mixture incubated at 60°C. After centrifugation (10 min, 3000 rpm) the aquaous layer was extracted with 5 ml chloroform, collected and after addition of 1/3 volume of 8 M LiCl, RNA was allowed to precipitate overnight at 4°C. The RNA precipitate was collected by centrifugation at 4°C (10 min, 12000 rpm) and washed twice with 70% ethanol. The dried pellet was finally dissolved in 0.06 M phosphate buffer pH 6.5. Northern blotting 5 μl RNA (2μg/ul) is denatured in 5 μl glyoxal and 10 μl DMSO for 60 min at 50°C and separated according to length on a 1% agarose gel (3.5 h, 40 Vj in 15 mM phosphatebuffer. Subsequently RNA is blotted to Hybond N membrane according to the manufacturer (Amersham) . The blot was prehybridized and hybridized following the procedure recommended by the membrane manufacturer. The probe (MS2 cDNA) was [ 32 P] random prime labelled. 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Claims: CLAIMS 1. A gene isolated from the cDNA library of a plant which encodes a protein indicated as MS2 which is involved in pollen development, said gene being characterized by the nucleotide sequence : sr rsa err AAS err err rcr aar SIA esa sas scs ror -roc rά. sea CCA J ά err crά -i Hat Sla Ala __.« 7&a Las Sax l«r Sac Sax Sa_c Sac Xla Taj. sly Sax Aaa Lya Lao 31 ZO ASS CAS ocr ere rrc rrs ACT res rcr rcr re: res ASS SSA SSS rca. AAC AAS err ACS «o λx? ma j o κia cya v« rrp Jar rs? val iia Ar? An Lya Lya Ar? ϊ a aiy rra 40 ASC CAC AAC or ror ore es rcr ACA SSS Arr AHA sir AAS AAA ACS rre ocr ecc iso Cya Ar? Val Sly Gly Sly Si-' n Gly Cly Art; Aaa Aaa Ala Cla Sar Pro 10 rsc csr era ssr ssr sci ear SAT ecr sss ACA AAC ACT AAC SCA SAC ACT eer - a Ax? VaJ. Sa Sax α Las Lya Are Ax? S17 Sla 7S_i Laa Xla Ax? Sla Ola 5ax Sax 10 AT? ess srr rcr res err rrs AAA SAC ACA SET cac ssa ers ASS ASS SU CAS ACT res z«o ?xo Ala M c Asp Ala Sla S___r Laa 71 Laa A fro Asa Sly Aaa Oly Ax? r__ιx Xla Sla 100 ere scr ASS SAT scr SAS ACA rrs srr ere rcr CCA AAC SSS AIT ssα ACA ACS ASS SAC 300 Ii Aaa Slv 7*1 Lya sir Las Mac Tr yfca Sax Slv al■■ j r Mac val ly Hat Lya sla 1= Arc AAT OCA ST AAS Acr rrs Arc ecr sss ACT see scs rcr ATO OIC SSS A=S AAA SAX 3*0 1 c "-'f -*° 61 / —•* —• jΛr ">• Laa Cla Sly lya Lya Jba Laa Xla rhr Oly Sax rtr Sly 140 — -> ϋ err ssc ASA ATC ACT rrc ere =-i sss AAC AAS rrr esA ASC ACT SSC res AC= ssr ca ?-a Laa Lya Val __*n Xla Sla lyi V*l Laa x? Mat Ala Pro Are Val Sax Lya Xla SO rrc rsa scr AAA SIA ere Arr SAS AAA arc rrs ACA rc ecr err SAT ore AGC AAS ASA <ao Syr Lai Laa Xla Lya AL* Lya Sax Lya Sla Ala Al* 11a da Ar? Laa Lya Aaa Ola al ISO CAT ere rrs ass AAA s e AAA A C AAA SAA αer scs Ar OLS ess csa AAC AAC SAS am 340 Laa Are Ala Sla Laa Pha Aaa r___ aa Lya Sla Tir »1 - Sly Ala Sax ryr M e Sax I&a 200 rsa. SAC CCA SAC err rrr AAT ACT csa AAA SAS ACT CAT cα OCA rcr sac rc rcr rrc t o 2 0 M c Sly Aaa Ua era Aaa Sax Aaa Xla Sly Laa Ola 2T0 ATS SSA AAC ATT SEC EC TCA AAC ATT SSS ITS OΛ. « β AlA A_rp Ala Sla Ola Ua AJj. > Ola 7 L A_ra Vail Ua _S.a Aaa Sat AX* » ' • Aaa Z40 seA SAT rca. scr EAA sac Arr see AAA SLA err az sir ACA ATC AAS rcr scr scr AAI 720 ri ^__ rb Aaa Sla x? ?τr Axp Va Ala Laa Arp Xa Aaa r_l_r AT 7 Oly fro Sly Aaa 2S3 ACA ACT rrc AAT SAA ASA ran SAT srr scr ers SAC ATC AAC ACA ACA SSS cec sss AAT 7«O Laa Mat Sly Tha ALa Lya Lya Cya Lya Lya Laa Lya Laα ba Laa Ola Val Jar S&r Ala 2<0 _ r - ere ΓS so. rre see AAC AAC SSC AAC AAA. ere AAA ers rrc rrs c OSA sec ACA ser t4β Tγ-: v_ι_l Aaa Sly αl_a Arq- Sla Sly Ax? Xla Mat Sla Lya fro r___a Sax Jtat oly Λxp cya 300 rar σrs AAT SSA CAA ACA CAA SCA ACE ASC ASS sac AAC C=A rrr rcr ASS SEA sas ror 900 Xla AXa ≥r Ola Aaa Iha Laa Sla Slτ Aaa Axir Lya AJ-a. Laa Aap VaJ. Aap Arq- Sla Mac 329 A=A s a ACA sac AAC rre ere SAA aa AAC ACA AAA CCA rri sa_r sir sas ACA sac Ara 9«o ys Laa Ala Laa Ola Ala Ala. A? Lya Sly Sr Sla Aaa Sla Are Ola Ala. Sla. Lya Mat 340 AAC rra. scr err saa scr scr ACA AAA sαα ACT CAA AAS U car sac sea cs AAS ASS 1020 Lya AT ? Laa sly Laa Sla Ax? Ala. Ax? Sax Sy? Sly r_rp "i- Are t___x T ? VaJ. Tha Sir 360 30 Jua ∞ ere cas era sac ess oca ad SCA sas sea rsc ■-" oc ACT ras orr rrc ace 1010 3S0 1140 400 1230 Ax-qr Mat Mat Asp Vrα Xla a Laa Cra T Gy Lya Oly Sla Laa Sir Oly !&a Laa Val 420 ACS ASS ASS sas ccs asa ssr rra. rsr sac sss AAS SSS CAA CΓC ACS SSS SSS rrs srr 12so 5 Λπ> ^ ^T Sir "al Laa Are aJ. v txo Ala Aaa Mat Val 'Val. Aaa AJ-a S&r Laa Ala 440 ear cca. AAA SSA err ers sas SSA ore e=s ses oi jaa orr αss AAS sea. ACS ssa. scr uzα Ala Xla Ala Lya H_ sly Mat Ala Mat t^x re fro sla Ira da Xla Aaa VaJ. -7? Sla 4 SO scr ASA cca AAC cas ssa ASS sea. ASS sea. SAS ees SAA ees SAA ASA AAC SSS sas aa 13S0 Xla Ala Sax Sax Ala Xla Aan fro Laa V*l Tha Ola Are Laa Ala Ola Laa Laa Syr Aaa 410 ASC scr rcr res scs ASA AAC CCS ess err ssc SAA S rra. scs sas err ss sas AAC 1440 E a Syr Lya Sir Sax Pro Cya Mae Aaa Lya Oly Aap trα Xla Mat v _l x? Laa Mat S00 eac si AAA ACA sec eca. soc ASS sac ses AAA SSS sas ess ASS ASS σiα ess sss ASS I_SOO 40 Lγa Laa ffca Aaa Val Are Aap Jta Are Kla Laa Srp Ax? Aap Ala. Sla Sla Ax? 529 AAA ess rrc AAS sec sss si sas ss: scs SAS car sss sss "?» sas scs ca saa. csα is s Sar Sly Laa Mat Sax Sly Mat Sar A a Aap Lya Mat Mat m - Lya Laa Lya Tba 540 acr ssc rrs ASS ASS SSS ASS ACT Γ=. SCS sas ACT AAC ASS ASS αc AAS era. AAC rrs £2o Xla cya Lya Lya Sar Va Sla Sla Ala Lya Sla Laa » ' - r≥x Xla Syr Sla Pro ?7T S_x 360 ASA SSC AAS AAA SCS SSS SAA CAA OCC AAA CSC CSS SCS ACT ATT SAS SAC CCA SAC ACT 1£S0 7 » ryr Sly Sly Ax? r Aaa Aaa s^x Aaa ^ = Sla Ax? Laa abac Ola Aaa Mat sla 580 ύ.5 rsc sas ssr SSA ACA rrs sas AAC ASC AAS ACA cac ACA SSA A=S sac AAS ASS sea. oc i"<° Are Sla Lya Ar? Sla 7h» Sly Fba Are Va sly Sar Xla Aaa ^p r__ιr Ajrp ryr Xla —— COO SAC SAC AAC ASA SAA rSS SSA SSS Sas SSS SSA ACC ASS AAC SSS ACS SAC 3C ASS ACA 1SO0 Aaa a E a Xla Ira Sly Laa Ax? Arr HJ-a Tal Laa Lya Sly Ax? Ala • £20 AAC srr cac ASS ecc scs rsx ACA ASS cas sre rrs AAA SSA ACA SCS SAA err ssa asc nβo rca. crx AAC cac ace AAA aa AAS cca rcc err err rra. rcr rrr ras ess srr err rrr iszo res. rra cer ssa ASC see rrs rcr era ASA SAS cac esc sas rre raA SAA rrr SAA AAA isβα AAC ess AAA rsr rsr sas ers raA srr rsc cca. AAA rcr ASA ore ass-rrc sas sea ACA 2040 CAA AAA AAA AAA AAA- J' 2925 50 or a homologous gene 2. A promotor of the S2 gene as defined in claim 1 isolated from the chromosomal DNA of a plant, said promotor being characterized by the nucleotide sequence: ) GATCTAAGACAAAAACGTCGCCATTTGCTAATTG1ll_1'ii'llU'liGTAGCAATAACCTTA 60 GTCAAAGGAXXTTGTTTATTGCGGACCCAAGTTGGTTGGTCGGCTCTTGCTIAAACCACA 120 TTTGGAATrTGTTGTTCTGGACTCTGGAGATCATTCAAACACAACCAAGAAGATACCCCA 180 CTGGT TTAAAGTCGTATGTGTAm1Cl rCT'l'CACCACGAGTTTAAGGTTGTCTTTCAT 240 GTCTCATTGTTCTAJATATTCATCTTCGGTTGCATGTTTAACTTCATACTCCAGTTTATA 300 TTTTCCATCTAGATGArrGGGAACATTTTGCITA ϊITXAIGATCTTAAACAGATGAACG 360 GTCXCAXGTTAACAACATAGTACTCTTGACTTCATGAIAATTTCATATCATCTAATGACT 420 AAATTCTTTGCAGAGTrTAAIGGTGTTGATTGTTGAAACAAGAGCAGATXGGTCAATCAC 480 ^CAG AAAAAAAACri CCTAAC^TrrAACrTTTA CGTT TTT ^ 540 AGT'm.CTACAAAACCTATAA'l1 lATGATGACCATATAATCCTCAAACCCTTCAAGAT 600 GTGATGTGAATTATCTAAATCCCAAC^CGAA^^TGACAITn'TlAAAGTTAGClATTT 660 ATCCTTAGTTGA' 1 llAATTATAGGGTAATGGCAA-rAl1 GGAACTGAIAATACG 720 iιn.iiiiiiiιiiCIGAATXCIAGATGATCA GTGTAGGAAACTGATAAAATGTTGGAA 780 AGAATTCGTAAGGCAATCII 'lATrTCACTTGAl111lAAAATATTTATTTGCCTAIAAA 840 0 ACAGAGGAAGTTTTTCAXCATCTTTTGTCCTTAGAACTAACCAATCTrTCArCCTCTTA 900 *5' cDNA XAAAAACAAAACCTACTTTAC XGTCTCTTAACGATAACAAAATAACAAATAATXAATTC 960 TGTTCrTGGTTrACXTAATCTTCTTTCTAGTTAAGTATATTCTTGTTGCTCATCACCAAA 1020 GGTATGCTTTC AGCTTAAGTATAT ACAAGTCACCAATTTCTTAACCAACAAGCTT 1077 0 5 or a homologous promotor. 3. A recombinant polynucleotide which can be suitable used for obtaining a male-sterile plant, essentially comprising: a) an inhibitor gene capable of inhibiting the expression of a target gene, present in said plant, encoding the MS2 protein involved in pollen development, said gene having the sequence as defined in claim 1 or a homologous target gene, and b) a promotor that is active in the tapetum of said plant, operably linked to said inhibitor gene as to achieve expression thereof in the tapetum of said plant. 4. A recombinant polynucleotide according to claim 3, wherein the inhibitor gene is a sense gene or an antisense gene. 5. A recombinant polynucleotide according to claim 3, wherein the promoter that is active in the tapetum of a plant comprises the promoter of the S2 gene or a fragment of the MS2 promotor showing the same expression specificity, said promotor having the sequence as defined in claim 2. 6. A method for obtaining a male-sterile plant, comprising the steps of a) transferring a recombinant polynucleotide according to any one of the claims 3 to 5 to cells of a male- fertile plant, b) generating whole new plants from cells having incorporated said recombinant polynucleotide, and c) selecting a plant that is male-sterile. 7. A recombinant plant genome, comprising incorporated therein a recombinant polynucleotide according to any one of claims 3 to 5. 8. A male-sterile plant comprising the recombinant plant genome of claim 7. 9. A cell, fruit, seed or progeny derivable from a male- sterile plant according to claim 8. 10. Self-fertilized seed of a male-sterile plant according to claim 8. 11. A homozygous male-sterile plant obtainable from a seed according to claim 10. 12. A cell, fruit, seed or progeny derivable from a seed according to claim 11. 13. A method for the production of heterozygous male- sterile plants by fertilizing a homozygous male-sterile plant according to claim 11 with a male-fertile plant, harvesting the seed and growing heterozygous male- sterile plants from said seed. 14. A method for obtaining hybrid seed comprising the steps of crossing a male-sterile plant from claim 8 or 11 with a male-fertile plant and collecting the hybrid seed.

Inventors: AARTS MARK GERARDUS MARIA, PEREIRA ANDY, STIEKEMA WILLEM JOHANNES, AARTS, MARK, GERARDUS, MARIA, PEREIRA, ANDY, STIEKEMA, WILLEM, JOHANNES
Applicants: AARTS MARK GERARDUS MARIA, CPRO DLO, PEREIRA ANDY, STIEKEMA WILLEM JOHANNES, AARTS, MARK, GERARDUS, MARIA, CENTRUM VOOR PLANTENVEREDELINGS- EN REPRODUKTIEOND, PEREIRA, ANDY, STIEKEMA, WILLEM, JOHANNES
Application year: 1994
Main classification: C12N15
IPC codes: C07K14, C12N15